This invention relates to the isolation and purification of polypeptides produced using genetic engineering techniques. More particularly, it relates to the design of genetically engineered fused polypeptides and the isolation and purification of target polypeptides therefrom by cleavage.
Advancements in recombinant DNA technology permitting foreign genes to be incorporated into various cells have made possible the expression of a product foreign to the cell. The molecular biology governing the expression of recombinant DNA places a variety of constraints on the form of polypeptides or proteins produced in the host organism. For instance, intracellular enzymes will often degrade the protein of interest, and it may be difficult to separate from other materials expressed by or comprising the structural materials of the host organism.
The development of fusion or hybrid polypeptide methodology has facilitated the protection, isolation, and purification of genetically engineered polypeptides and overcomes some of these constraints. Protection from intracellular degradation can be accomplished by fusing a sequence of amino acids to the target protein to avoid digestion by enzymes within the cell. A fusion product can be engineered to be secreted from the host into the growth medium by fusing a signal peptide to the target. In addition, the hybrid can be designed to facilitate isolation and purification if the desired protein is fused to a polypeptide having characteristics exploitable in purification. The fusion product is prepared by engineering the gene coding for the protein of interest in reading frame with a DNA sequence of a polypeptide other than the protein of interest. For instance, DNA specifying a positively charged peptide or other molecule can be fused to the DNA encoding the target protein, allowing the protein product to bind strongly to negatively charged ion-exchange or affinity matrices.
The production of proteins with fusion methodology is also faced with constraints. For example, the material fused to the target protein must not permanently interfere with the biological activity of the target protein, and efficient cleavage of the fused material to obtain purified target protein can be difficult to achieve. Often only some fraction of the fusion protein is properly cleaved, and therefore existing methods provide low purification efficiency. Furthermore, the cleavage agent may cleave at several locations resulting in product loss and the production of contaminating fragments.
Fusion methodology has been discussed at length in the prior art. For example, European Patent Application No. 0047600 is understood to disclose a process for synthesizing bovine growth hormone by producing a fusion protein and purifying the growth hormone from the culture medium of the host organism. Other proteins have been produced through fusion techniques.
Generally, the prior art teaches that genetic material encoding a cleavage site can be incorporated between the DNA encoding the desired protein and the DNA encoding the additional fused material. Expression yields a precursor protein comprising the amino acid sequence of the target polypeptide linked to one or more peptides defining a selected cleavage site and another amino acid sequence. EPO 0035384 discloses the use of fusion proteins having specific cleavage sites incorporated in their structures which facilitate purification of expression products. EPO 0161937 discloses a recombinant fusion protein including a DNA sequence encoding a site specifically cleavable by blood coagulation Factor Xa. EPO 0163573 is understood to disclose a nucleotide sequence inserted into a plasmid expression vector which codes for an amino acid sequence specifically recognizable by the proteolytic enzyme renin.
The fusion product can comprise a moiety designed to facilitate isolation of the desired product. For instance, PCT/84/03103 discloses a DNA sequence coding for the protein of interest linked to a DNA sequence coding for protein A or another macromolecule capable of binding to the constant region of immunoglobulins. The fusion protein is purified by adsorption of protein A to immobilized IgG. In addition, a cleavage site can be incorporated between the protein A fragment and the protein of interest. The target protein can then be cleaved from the remainder of the fused product adsorbed to the IgG carrier. U.S. Pat. No. 4,431,739 also describes a method of expressing a precursor protein containing a desired protein, an additional polypeptide, and a selective cleavage site adjacent the desired protein.
Recombinant technology potentially can provide useful proteins in large quantities in easily purifiable form. Large quantities of pure proteins are needed for clinical use and research. Therefore inexpensive, efficient, high yielding production methods are necessary. What is needed is an efficient method for the purification of polypeptides produced through recombinant DNA which is generally independent of the properties of the cloned protein. This would enable adaptation of the procedure to the production of any desired protein. What is also needed is a method for increasing the yield of the purified proteins made using the fusion technology.
It is an object of this invention to provide a procedure for the production, isolation, and purification of genetically engineered proteins. Another object is to provide a method for obtaining recombinant protein of interest in improved yields. Another object is to provide a method which can be adapted to any polypeptide of interest which can be coded for and expressed by a host organism. Still another object is to provide such a procedure which is both efficient and inexpensive.
These and other objects of the invention will be apparent from the description, drawing, and claims that follow.